CN107075504B - Schwann's cell and its preparing process - Google Patents

Abstract

The present invention addresses the problem of providing a method for directly obtaining Schwann cells (by direct reprogramming) without using pluripotent stem cells such as ES cells or iPS cells. As a means for solving the above problems, a method for producing schwann cells is provided, which comprises a step of introducing at least 1 gene selected from the group consisting of the SOX10 gene and the KROX20 gene, or an expression product thereof, into somatic cells of a mammal.

Description

Schwann's cell and its preparing process

Technical Field

The present invention relates generally to schwann cells and a method for preparing the same, and more particularly, to a method for preparing schwann cells by direct reprogramming.

Background

Schwann cells are thought to play a crucial role in the regeneration of nerves. There are many diseases associated with neurological deficit and functional deficiency of schwann cells, and it is expected that these diseases will become ideal regenerative medicine if schwann cells can be transplanted. In fact, the treatment effect of transplantation of an autologous nerve or transplantation of schwann cells isolated and cultured from an autologous nerve is improved in the case of nerve damage caused by trauma or removal of malignant tumor. However, the collection of nerves is highly invasive to the patient and secondary nerve damage cannot be avoided anyway. In addition, the number of Schwann cells that can be supplied is often insufficient.

Non-patent documents 1 and 2 disclose the following methods: differentiation of schwann cell-like cells (dADSC) was performed using mesenchymal stem cells as a material, which are represented by undifferentiated adipose-derived stem cells (ADSCs) (also referred to as adipose-derived stromal cells). However, since the nature of the method involves a risk of infection from the outside, there are problems that quality control is not easy, and cost and labor are required. Further, it is also pointed out that: the obtained cells are different from true Schwann cells in shape and function. Further, it has not been reported that Schwann cells produced by these methods have a myelin ability and may not contribute to hopping conduction.

Recently, it has been shown that cardiomyocytes or hepatocytes and the like can be directly induced (directly reprogrammed or directly converted) by fibroblasts. If schwann cells can be directly produced from somatic cells such as fibroblasts collected from a patient with low invasion, the invasion is low and the risk of canceration is low, and a new technique for producing autologous schwann cells for transplantation is expected.

The following reports have been made, for example, on the fact that a genome into which a tissue-specific transcription factor is introduced into a somatic cell can be directly differentiated and induced into the somatic cell without passing through iPS cells (direct reprogramming (direct conversion)).

Mouse fibroblast → chondrocyte (introduced SOX9+ Klf4+ c-Myc gene)

Mouse fibroblast → myocardial cell (introduced GATA4+ Mef2c + Tbx5 gene)

Mouse fibroblast → hepatocyte (Hnf 4. alpha. + (introduced Foxa1 or Foxa2 or Foxa3) gene)

Mouse fibroblast → neural stem cell (introduced with Sox2+ FoxG1 gene, etc.),

Mouse, human cells → hematopoietic stem cells, etc.

However, there is no report of direct conversion of somatic cells into Schwann cells.

Documents of the prior art

Non-patent document

Non-patent document 1: kingham PJ, DF Kalbermanten, D Mahay, et al, adopise-derived stem cells differential intro a Schwann cell phenotype and baromote neural output in vitro, 2007; 207:267-274.

Non-patent document 2: liu Y, Zhang Z, Qin Y, Wu H, Lv Q, Chen X, Deng W A new method for Schwann-like cell differentiation of adipose derived stem cells Neurosci Lett.2013Sep 13; 551:79-83.

Disclosure of Invention

Problems to be solved by the invention

An object of the present invention is to provide a method which can be applied to the treatment of diseases associated with neurological deficit and functional defect of schwann cells, and to produce schwann cells with a low risk of canceration.

Means for solving the problems

The invention discovers that: when a specific gene is incorporated into a somatic cell of a mammal, a schwann cell can be obtained directly (by direct reprogramming) without using a pluripotent stem cell such as an ES cell or an iPS cell.

The present invention includes the following aspects.

Item 1, a method for producing a Schwann cell, comprising a step of introducing at least 1 gene selected from the group consisting of the SOX10 gene and the KROX20 gene, or an expression product thereof, into a somatic cell of a mammal.

Item 2 the method of item 1, wherein the gene is a combination of the SOX10 gene and the KROX20 gene.

Item 3, the method of item 1 or 2, wherein the somatic cell is a fibroblast, a vascular endothelial cell, or a mesenchymal stem cell.

Item 4, a schwann cell derived from a somatic cell of a mammal, having at least 1 gene selected from the group consisting of an exogenous SOX10 gene and a KROX20 gene, or an expression product thereof.

The transplant material according to item 5 for treating a disease caused by a defect in a nerve or a defect, deficiency, or decreased function of Schwann cells, which comprises the cells obtained by the method according to any one of items 1 to 3 or the Schwann cells according to item 4.

Item 6, a composition for preparing Schwann cells, which comprises at least 1 gene selected from the group consisting of the SOX10 gene and the KROX20 gene, or an expression product thereof.

Effects of the invention

In the present invention, schwann cells can be supplied from somatic cells in a short period of time by direct reprogramming. Since the schwann cells can be easily induced by the somatic cells of the person who is transplanted, the obtained schwann cells do not cause problems such as immunological rejection response when transplanted. In addition, since schwann cells can be directly induced from somatic cells without passing through iPS cells or ES cells, problems caused by pluripotent stem cells such as canceration can be avoided.

Drawings

Fig. 1 shows an outline of an example of the method of the present invention.

Fig. 2 shows a representative stainability image of S100 β.

Fig. 3 shows a typical example of 4 stages of S100 β obtained by dyeing property.

Fig. 4A shows the results of evaluation based on cell morphology. Typical examples of cell morphology of HDF, cssc, and dSC are shown.

Fig. 4B shows the results of the evaluation based on the cell morphology.

FIG. 5A shows an example of immunostaining for a Schwann cell-associated marker (p75 NTR).

Fig. 5B shows an example of immunostaining for schwann cell associated marker (GFAP).

FIG. 5C shows an example of immunostaining for a Schwann cell-associated marker (Nestin).

FIG. 5D shows an example of immunostaining for a Schwann cell-associated marker (NG 2).

FIG. 6 shows the results of measurement of mRNA expression levels of S100. beta. and p75NTR genes.

Fig. 7A shows the results of evaluation of neurite elongation effect on nerve cells. One example of fluorescently labeled NG108-15 nerve cells co-cultured with each of fibroblast HDF, normal Schwann cells (cSC, positive control) and dSC is shown. Arrows indicate elongated neurites.

Fig. 7B shows the evaluation results of the neurite elongation effect on nerve cells.

FIG. 8 shows an example of immunostaining for Schwann cell-associated markers (S100. beta., p75NTR, GAP43) during transformation by Plasmid introduction (electroporation).

Fig. 9A shows a phase difference image of human normal adipose-derived stem cells (ADSCs) before Induction, a phase difference image after Induction of schwann cells, and a staining property image of S100 β. Magnification is 200 times.

FIG. 9B shows an example of immunostaining for Schwann cell-associated markers (S100. beta., GAP43, p75NTR, Protein Zero (PO)). Magnification is 100 times.

Fig. 10A shows a phase difference image of umbilical vascular endothelial cells (Huvec) before Induction, a phase difference image after Induction of schwann cells, and a staining property image of S100 β.200 times.

FIG. 10B shows an example of an immunostaining image of Schwann cell-associated markers (S100. beta., GAP43, p75 NTR). Magnification is 100 times.

FIG. 11A shows an example of an immunostained image of a myelin marker (Protein Zero (P0)).

FIG. 11B shows an example of an immunostained image of a Myelin marker (Myelin basic protein (MBP)).

Fig. 12A shows the outline of the evaluation test using the sciatic nerve injury model.

FIG. 12B shows an example of an immunostained image using a marker associated with Schwann cells.

FIG. 12C shows an example of an immunostained image using a marker associated with Schwann cells.

Fig. 13A shows the outline of dSC transplantation test for the immunodeficient mouse ischial defect model.

Fig. 13B shows a macroscopic image of the nerve reconstructed.

FIG. 13C shows a myelin stained image of a transverse axial section of regenerated nerve tissue.

Fig. 13D shows the evaluation results of sciatic nerve function index (SFI).

FIG. 13E shows the results of the evaluation of atrophy and fibrosis of Innervated muscle.

FIG. 14 shows the production of neurotrophic factors (BDNF, GDNF, NGF). The measurement was carried out by ELISA method. P <0.05vs. control; p <0.01vs. control; # p <0.05vs. cSC.

Fig. 2, 3, 4A, 5A to D, 7A, 8, 9A and B, 10A and B, 11A and B, and 12B to C simultaneously show a color reversal image.

Detailed Description

The invention relates to a preparation method of Schwann cells. The method of the present invention is a method for producing a schwann cell without using a pluripotent stem cell such as an ES cell or an iPS cell.

Schwann's cell

Schwann cells are glial cells in the peripheral nervous system. Under physiological conditions, it is helpful to realize skip conduction by supporting nerve tissues and forming myelin (marrow sheath). In peripheral nerve injury, many important roles in peripheral nerve regeneration, such as production and release of neurotrophic factors, regeneration of the base of axons, and myelin formation, are performed.

Natural schwann cells originate from the nerve ridge, unlike nerve cells that originate directly from the ectoderm. Mature Schwann cells are formed by Schwann precursor cells and immature Schwann cells. For simplicity, all cells in the middle of these differentiation processes are included in the term "schwann cells" in the present specification.

In addition, in the schwann cell, there are schwann cells that form myelin, migratory schwann cells that do not form myelin (undifferentiated schwann cells), and the like, and all of these are included in the "schwann cells" in the present specification.

In the present specification, cells that include not only the same cells as natural schwann cells but also a part or all of the functions of the cells are considered to be the same cells as natural schwann cells (may also be referred to as "schwann cell-like cells"), and are referred to as "schwann cells".

Somatic cell

The somatic cell may be derived from a mammal. Particularly preferred are cells derived from mammals, which are not Schwann cells themselves or cells having the ability to differentiate into Schwann cells in vivo. In the case of transplanting schwann cells into a living body, it is preferable to use somatic cells (self-cells) derived from a subject to be transplanted in order to reduce the risk of infection, rejection response, and the like. However, in the case of transplantation for the purpose of, for example, sudden nerve damage, etc., schwann cells prepared in advance from somatic cells of another person or other animals, rather than self cells, may be used for transplantation. Or, schwann cells can be prepared from somatic cells of another person or other animal prepared in advance and used for transplantation. That is, a schwann cell bank (a bank containing schwann cell precursor cells) can be prepared and used for transplantation purposes. In this case, in order to reduce the risk of rejection response and the like, MHC may be classified in advance. In addition, the cell characteristics, tumorigenicity, and the like of Schwann cells can be confirmed in advance.

In the present specification, examples of mammals include mice, rats, hamsters, humans, dogs, cats, monkeys, rabbits, cows, horses, and pigs, and particularly, humans are mentioned.

The somatic cell to be subjected to the method (direct reprogramming) of the present invention is not particularly limited.

As the somatic cells, somatic cells that can be easily collected from an organism can be used. Examples thereof include: fibroblasts, keratinocytes, oral mucosal epithelial cells, nasal mucosal epithelial cells, tracheal mucosal epithelial cells, gastric mucosal epithelial cells, intestinal mucosal epithelial cells, vascular endothelial cells, smooth muscle cells, adipocytes, gingival cells (gingival fibroblasts, gingival epithelial cells), tooth marrow cells, tooth root membrane cells, bone marrow-derived mesenchymal cells, leukocytes, lymphocytes, muscle cells, conjunctival epithelial cells, osteoclasts, and the like, and preferably fibroblasts, keratinocytes, oral mucosal epithelial cells, gingival cells, leukocytes, lymphocytes, and the like. In the present invention, it is preferable to use the above-mentioned cells collected from a living body.

"organism" includes not only embryo (fetus), larva, and adult, but also placenta and umbilical cord connecting mother and fetus. Umbilical cord vascular endothelial cells and the like umbilical cord-derived cells or placenta-derived cells are not strictly speaking somatic cells, but these cells are also included in the "somatic cells" of the present invention (in this case, "somatic cells" are read as "umbilical cord vascular endothelial cells", "umbilical cord-derived cells", "placenta-derived cells" and the like by another phrase). From the viewpoint of ease of collection, these cells are one example of preferable somatic cells.

Examples of somatic cells include somatic cells prepared by inducing differentiation, dedifferentiation, or reprogramming somatic stem cells such as Mesenchymal Stem Cells (MSC), Neural stem cells (Neural stem cells), hepatic stem cells (hepatic stem cells), intestinal stem cells, skin stem cells, hair follicle stem cells, and pigment cell stem cells. Further, cells induced into other somatic cells by differentiation, dedifferentiation, or reprogramming from various somatic cells are also included. In addition, somatic cells induced by differentiation induction, dedifferentiation, or reprogramming of germ line cells are also included.

In addition, somatic cells induced by differentiation or reprogramming of Embryonic stem cells (ES cells) or artificial pluripotent stem cells (iPS cells) are also included.

In addition, somatic stem cells may be used in addition to the differentiated somatic cells described above. Herein, the stem cell refers to a cell having a self-replicating ability and an ability to differentiate into other types of cells. Specifically, the following can be exemplified: mesenchymal Stem Cells (MSC) (for example, adipose-derived Mesenchymal cells (ADSC)), Neural stem cells (Neural stem cells), hepatic stem cells (hepatic stem cells), intestinal stem cells, skin stem cells, hair follicle stem cells, pigment cell stem cells, and the like.

In addition, not strictly speaking, a somatic cell, and an ES cell, an iPS cell, or a germ line cell are also included in the "somatic cell" of the present invention (in this case, the "somatic cell" is read as "ES cell", "iPS cell", or "germ line cell" by the other words).

Further, cultured cells may be mentioned, and somatic cells induced by differentiation induction, dedifferentiation, or reprogramming of the cultured cells may be mentioned. In addition, somatic cells induced by differentiation, dedifferentiation, or reprogramming of ES cells, iPS cells, or germ line cells are also included.

In a preferred embodiment of the invention, the somatic cell is a fibroblast, a vascular endothelial cell (in particular, a vascular endothelial cell of umbilical cord) or a mesenchymal stem cell (in particular, a mesenchymal cell derived from fat).

Genes or expression products thereof

In the method of the present invention, at least 1 gene selected from the group consisting of SOX10 gene and KROX20 gene, or an expression product thereof, is introduced into a somatic cell. Here, the "expression product" may be mRNA or protein of SOX10 gene and/or KROX20 gene.

The combinations of genes that can be used include a combination of only the SOX10 gene, only the KROX20 gene, the SOX10 gene, and the KROX20 gene. From the viewpoint of the efficiency with which schwann cells can be obtained, a combination of the SOX10 gene and the KROX20 gene is preferable.

In the method of the present invention, a gene other than 1 or 2 of the SOX10 gene and/or KROX20 gene may be used simultaneously. In addition, microRNA, siRNA, shRNA, or DNA expressing them may be used together. In addition, various proteins may be used together. In addition, it may be introduced together with various other genes. From the viewpoint of efficiency with which schwann cells can be obtained and from the viewpoint of simplicity, it is preferable to use only 1 or 2 kinds of genes, in particular, two kinds of genes of the SOX10 gene and the KROX20 gene.

The SOX10 gene encodes a transcription factor belonging to the SOX (SRY-related HMG-box) family and involved in the control of the determination of cell fate in the occurrence of embryonic stage.

The KROX20 gene (alias, EGR2, AT591, CMT1D, CMT4E) encodes a protein with 3 zinc fingers (zinc fingers) of the C2H2 type.

The above genes are highly conserved in vertebrates, and in the present specification, unless an animal name is specifically indicated, they indicate genes including homologues. Further, the gene includes a gene containing a polymorphism and having a function equivalent to that of a wild-type gene product even if it is a mutated gene.

For example, the cDNA nucleotide sequences of the SOX10 gene and KROX20 gene of human (Homo sapiens) and mouse (Mus musculus) and the amino acid sequences of the proteins encoded thereby are registered (in the case where a plurality of revisions are registered, they are understood to mean the latest revision) in GenBank provided by the National Center for Biotechnology Information (NCBI) under the following Accession Number:

human SOX10 gene cDNA sequence: NM-006941 (e.g., NM-006941.3),

Human SOX10 protein amino acid sequence: NP _008872 (e.g., NP _ 008872.1);

mouse Sox10 gene cDNA sequence: NM-011437 (e.g., NM-011437.1),

Mouse SOX10 protein amino acid sequence: NP _035567 (e.g., NP _ 035567.1);

human KROX20 gene cDNA sequence: NM _000399, NM _001136177, NM _001136178, NM _001136179 (e.g., NM _000399.3, NM _001136177.1, NM _001136178.1, NM _001136179.1),

Human KROX20 protein amino acid sequence: NP _000390, NP _001129649, NP _001129650, NP _001129651 (e.g., NP _000390.2, NP _001129649.1, NP _001129650.1, NP _ 001129651.1);

mouse Krox20 gene cDNA sequence: NM _010118 (for example NM _010118.3)

Mouse KROX20 protein amino acid sequence: NP-034248 (e.g., NP-034248.2).

Introduction into

The method of the present invention can be carried out by a known direct reprogramming method, for example, a method according to any of the following documents, except that a specific gene is selected and a medium suitable for Schwann cells is used:

1:Direct Reprogramming of Fibroblasts into Functional Cardiomyocytesby Defined Factors；Masaki Ieda,Ji-Dong Fu,Paul Delgado-Olguin,Vasanth Vedantham,Yohei Hayashi,Benoit G.Bruneau,and Deepak Srivastava Cell 142:375-386,2010.

2:Direct conversion of fibroblasts to functional neurons by defined factors.Thomas Vierbuchen,Austin Ostermeier,Zhiping P.Pang,Yuko Kokubu,Thomas C.Sudhof&Marius Wernig.Nature 463:1035-1041,2010

3:Induction of human neuronal cells by defined transcription factors.Pang ZP,Yang N,Vierbuchen T,Ostermeier A,Fuentes DR,Yang TQ,Citri A,Sebastiano V,Marro S,Sudhof TC,Wernig M.Nature 476:220-223,2011.

4:Generation of hyaline cartilaginous tissue from mouse adult dermal fibroblast culture by defined factors Kunihiko Hiramatsu,Satoru Sasagawa,Hidetatsu Outan

i,Kanako Nakagawa,Hideki Yoshikawa,and Noriyuki Tsumaki,Journal of Clinical Investigation,121:640-657,2011.

5:Induction of functional hepatocyte-like cells from mouse fibroblasts by defined factors.Pengyu Huang,Zhiying He,Shuyi Ji,Huawang Sun,Dao Xiang,Changcheng Liu,Yiping Hu,XinWang&Lijian Hui,.Nature 475:386-389,2011.

6:Direct conversion of mouse fibroblasts to hepatocyte-like cells by defined factors.Sayaka Sekiya&Atsushi Suzuki.Nature 475:390-393,2011.

7 International publication No. WO2014/010746

The contents of the above-mentioned documents 1 to 7 are incorporated by reference in the present specification.

Specifically, it is preferable that the target gene is inserted into 1 or more expression vectors, and the expression vectors are introduced into the target somatic cells and expressed in the cells.

Examples of the method for gene transfer include a method of infecting a viral vector such as a retrovirus vector, adenovirus vector, lentivirus vector, adeno-associated virus vector, herpes virus vector, Sendai virus vector, and the like. In addition, when a gene and its expression product are introduced, a plasmid vector, an episomal vector, or an expression product (mRNA or protein) of the gene may be transfected with a non-viral vector such as cationic liposome, cationic polymer, or electroporation. Alternatively, mRNA may be introduced. These means for gene transfer are all included, and are referred to as vectors in the present specification.

From the viewpoint of the efficiency of introduction and the stable maintenance of the introduced gene, a viral vector is preferred, and a plasmid is preferred in order to suppress the risk of canceration.

Further, a gene to be a drug selection marker (puromycin resistance, blasticidin S resistance, neomycin resistance, hygromycin resistance, etc.) is introduced together with the target gene, and drug selection is performed thereafter, whereby a cell expressing the target gene can be selected and used.

The gene of the present invention can be introduced into a plasmid, or a viral vector, for example, a retroviral vector, can be used. From the viewpoint of the efficiency of introduction and the stable maintenance of the introduced gene, a viral vector is preferred, and a plasmid is preferred in order to suppress the risk of canceration.

The gene introduced into the somatic cell may be transcribed using the LTR promoter, or may be expressed from another promoter within the vector. For example, a constitutive expression promoter such as CMV promoter, EF-1. alpha. promoter or CAG promoter, or a desired inducible promoter can be used. In addition, a chimeric promoter in which a part of LTR is replaced with another promoter can be used.

When the transfer factor is an expression product (e.g., Protein) of a gene, a peptide called Protein Transfer Domain (PTD) or the like is bound to the Protein as the expression product and added to the medium, whereby the transfer factor can be transferred into a somatic cell.

In one embodiment of the method of the present invention, after introduction of a gene or the like into somatic cells, the introduced cells may be cultured in a medium suitable for culture of Schwann cells. As a medium suitable for the culture of schwann cells, a known medium can be used. For example, forskolin containing about 1 to 20 μ M (particularly about 5 μ M) in a normal Medium such as DMEM Medium (Dulbecco's modified Eagle's Medium) supplemented with 10% fbs (total bone serum) can be used; about 2 to 50ng/ml (particularly about 10 ng/ml) of bFGF (basic fibroblast growth factor); PDGF (Platelet-Derived Growth Factor) at a concentration of about 2 to 50ng/ml (particularly about 10 ng/ml); 1 or more (preferably all) of culture media (Schwann cell induction medium) containing 50 to 1000ng/ml (particularly 200 ng/ml) of human neuroegulin-beta 1 (alias, heregulin, GGF (Global growth factor)) or the like (the above concentrations are final concentrations). For example, the culture medium described in non-patent document 1 or 2 (a culture medium in which schwann cells can be induced from undifferentiated adipose stem cells) can be used.

The culture period is not particularly limited, and may be, for example, about 12 hours to 1 month, about 1 day to 3 weeks, or about 3 days to 2 weeks. If necessary, medium exchange may be performed. The culture conditions are preferably according to conventional methods.

Preparation of

In this way, Schwann cells were induced from somatic cells to prepare Schwann cells.

The prepared schwann cells have at least 1 gene selected from the group consisting of exogenous SOX10 gene and KROX20 gene or expression products thereof in a certain manner. Here, "exogenous" means a mode in which a gene or an expression product thereof is introduced mainly as a result of the above-mentioned introduction means, and a mode different from the natural mode. Examples thereof include a gene whose expression is controlled by a promoter other than a natural promoter, a position on a chromosome other than a natural promoter, and a gene present extrachromosomally.

The point that schwann cells are obtained, and the evaluation of the function of schwann cells and the like can be exemplified by: observation and evaluation of morphology (e.g., ratio of cell width to cell length); detection of expression of a marker specific to Schwann cells such as S100. beta., p75NTR, GFAP, Nestin, and NG2 (for example, detection of gene expression of a marker by RT-PCR method or the like, detection of expression of a marker protein by immunostaining or the like); production of neurotrophic factors; the effect of neurite elongation and the myelin-forming ability with respect to cocultured nerve cells.

Schwann cells typically have a bipolar or multipolar cell morphology with relatively small nuclei.

Among the schwann cell-specific markers, p75NTR is a marker for undifferentiated schwann cells.

The formation of Myelin can be confirmed by detecting a marker of Myelin cells such as Myelin Protein Zero (MPZ, P0) and Myelin Basic Protein (MBP), and observing the morphology of Myelin.

Schwann cells are sometimes obtained as a mixture with cells other than schwann cells (e.g., original somatic cells). In this case, the schwann cells and cells other than the schwann cells can be separated as necessary. Means for carrying out the separation is not particularly limited. For example, when the obtained schwann cells and fibroblasts that are the original cells are separated, the separation can be performed based on the difference in adhesiveness to cells of the ground substance (e.g., collagen). In general, schwann cells have poor adhesion to the ground as compared to fibroblasts. In addition, the schwann cells and cells other than the schwann cells may be separated according to classification.

The Schwann cells produced by the present invention can be preferably used as a transplant material described later, for example.

The schwann cells prepared by the present invention can be used for various studies and technical developments using schwann cells. For example, it is useful for basic studies such as analysis of the mechanisms of generation, differentiation, and morphogenesis of schwann cells, mechanical stress, nutrition, and influence of hormones on these factors.

Since the use of the schwann cells prepared according to the present invention enables the easy, rapid, and inexpensive establishment of schwann cells from humans or animals having various disease or genetic backgrounds, it is possible to analyze abnormalities in schwann cells associated with disease or genetic backgrounds by biochemical, molecular biological, immunological methods, and the like, and thus, it is useful for research such as elucidation of disease pathogenesis, and development of diagnostic methods. In addition, development of a drug, a toxicity test of a drug, and the like using such Schwann cells can be useful for development of a novel therapeutic method for various diseases.

Graft material

The Schwann cell obtained by the invention can be used for treating various diseases. In this case, schwann cells can be provided in the form of a transplant material.

The graft material is a material containing schwann cells, which is introduced into a living body for repair and reconstruction of nerve fibers. The Schwann cells obtained in the present invention can be used for producing a graft material. Schwann cells themselves also become the graft material. Therefore, the schwann cells may be transplanted into a patient as a cell preparation, together with a base material (scaffold) made of an artificial material, or after culturing together with a scaffold. The substrate (scaffold) functions, for example, as a nerve reconstruction. In these cases, the stent may be formed into various 3-dimensional shapes according to the purpose of transplantation.

The graft material of the present invention can be produced by a method including the above-described method for producing schwann cells as a step.

Specific examples of the base material (scaffold) include a polyglycolic acid (PGA) tube, a collagen tube, Fibrin glue (Fibrin glue), a polymer foam tube, an gelatin tube, polyglycolic acid (PGA), and a tube of collagen. As the tube in which polyglycolic acid (PGA) and collagen are combined, commercially available products such as "ナーブブリッジ" (manufactured by toyobo co., ltd.) can be used.

The transplantation material can be used for autologous nerve transplantation or for a treatment in which schwann cells are isolated from autologous nerves and cultured and transplanted. This method is described in the following documents:

1:Hadlock T,Sundback C,Hunter D,Cheney M,Vacanti JP.A polymer foam conduit seeded with Schwann cells promotes guided peripheral nerve regeneration.Tissue Eng 2000；6:119-127.

2:Jesuraj NJ,Santosa KB,Macewan MR,Moore AM,Kasukurthi R,Ray WZ,Flagg ER,Hunter DA,Borschel GH,Johnson PJ,Mackinnon SE,Sakiyama-Elbert SE.Schwann cells seeded in acellular nerve grafts improve functional recovery.Muscle Nerve.2014Feb；49(2):267-76.

3:Tabesh H,Amoabediny G,Nik NS,Heydari M,Yosefifard M,Siadat SO,Mottaghy K.The role of biodegradable engineered scaffolds seeded with Schwann cells for spinal cord regeneration.Neurochem Int.2009Feb；54(2):73-83.

4:Novikova LN,Pettersson J,Brohlin M,Wiberg M,Novikov LN.Biodegradable poly-beta-hydroxybutyrate scaffold seeded with Schwann cells to promote spinal cord repair.Biomaterials.2008Mar；29(9):1198-206.

5:Guest J,Santamaria AJ,Benavides FD.Clinical translation of autologous Schwann cell transplantation for the treatment of spinal cord injury.Curr Opin Organ Transplant.2013Dec；18(6):682-9.

6:Brook GA,Lawrence JM,Shah B,Raisman G.Extrusion transplantation of Schwann cells into the adult rat thalamus induces directional host axon growth.Exp Neurol.1994Mar；126(1):31-43.

7:Vaudano E,Campbell G,Hunt SP.Change in the molecular phenotype of Schwann cells upon transplantation into the central nervous system:down-regulation of c-jun.Neuroscience.1996Sep；74(2):553-65.

8:Keirstead HS,Ben-Hur T,Rogister B,O'Leary MT,Dubois-Dalcq M,Blakemore WF.Polysialylated neural cell adhesion molecule-positive CNS precursors generate both oligodendrocytes and Schwann cells to remyelinate the CNS after transplantation.J Neurosci.1999Sep 1；19(17):7529-36.

9:Wan H,An YH,Sun MZ,Zhang YZ,Wang ZC.Schwann cells transplantation promoted and the repair of brain stem injury in rats.Biomed Environ Sci.2003Sep；16(3):212-8.

10:Chen L,Fan X,Jin G,Wan X,Qiu R,Yi G,You Y,Xu Q.Treatment of rat with traumatic brain injury and MR tracing in vivo via combined transplantation of bone marrow stromal cells labeled with superparamagnetic iron oxide and Schwann cells.J Biomed Nanotechnol.2014Feb；10(2):205-15.

11:Shields SA,Blakemore WF,Franklin RJ.Schwann cell remyelination is restricted to astrocyte-deficient areas after transplantation into demyelinated adult rat brain.J Neurosci Res.2000Jun 1；60(5):571-8.。

the contents of the above-mentioned documents 1 to 11 are incorporated by reference in the present specification.

Examples of the disease to be treated include: defects or damages of central nerves caused by cerebral infarction, spinal injury, etc., defects or damages of peripheral nerves caused by trauma, or by removal of neuritis or tumor, etc.; central nervous system diseases such as multiple sclerosis, optic nerve cord inflammation (Devic syndrome), concentric sclerosis (Balo disease), Acute Disseminated Encephalomyelitis (ADEM), inflammatory wide sclerosis (Schilder disease), Subacute Sclerosing Panophantis (SSPE), Progressive Multifocal Leukoencephalopathy (PML); peripheral nerve diseases such as Guillain-Barre syndrome, Fisher syndrome, and chronic inflammatory hemorrhagic nerve diseases; Charcot-Marie-Tooth disease (CMT) and the like are based on diseases such as Schwann cell defect, deficiency or function reduction.

In the present specification, unless otherwise specified, the term "treatment" refers to a treatment performed during a period in which a patient suffers from a specific disease or disorder, and means to reduce the severity of the disease or disorder, or 1 or more symptoms thereof, or delay or deceleration of progression of the disease or disorder. In the present specification, "treatment" includes "prevention".

The Schwann cells obtained in the present invention are not limited to the treatment of diseases, and may be used for the purpose of beauty or function enhancement. In this case, the term "patient" may be used interchangeably with the term "healthy person" or "human" and the term "disease" may be used interchangeably with the term "beauty" or "function" with respect to human treatment, which is also referred to as treatment for convenience in this specification.

The present invention can be used not only for the treatment of human diseases but also for the treatment of diseases in mammals including pet animals such as dogs and cats and domestic animals such as cows, horses, pigs, sheep and chickens. In this case, the term "patient" is used as "affected animal" or "mammal".

Composition comprising a metal oxide and a metal oxide

As described above, schwann cells can be prepared by introducing at least 1 gene selected from the group consisting of SOX10 gene and KROX20 gene, or an expression product thereof, into somatic cells. Accordingly, the present invention further provides a composition for preparing schwann cells, comprising at least 1 gene selected from the group consisting of SOX10 gene and KROX20 gene or an expression product thereof. The composition for producing the schwann cell contains a factor used for inducing the schwann cell from the somatic cell, and the gene or the expression product thereof is preferably contained in a form that can be introduced into the somatic cell. The gene may be introduced into a somatic cell, and specifically, a vector into which the gene is inserted may be exemplified. Here, the genes may be inserted into different vectors, or 2 or more genes may be simultaneously inserted into 1 vector.

The kind of the carrier that can be used is as described above.

Direct reprogramming in vivo (in vivo)

As described above, schwann cells can be prepared by introducing at least 1 gene selected from the group consisting of SOX10 gene and KROX20 gene, or an expression product thereof, into somatic cells.

Here, when a nerve is injured, fibroblasts are collected at the injured part. The aggregated fibroblasts then form fibrous scars.

Therefore, at least 1 gene selected from the group consisting of SOX10 gene and KROX20 gene, or an expression product thereof, is introduced into fibroblasts in a damaged part of a nerve by applying the production method of the present invention, and schwann cells are induced in the damaged part by direct reprogramming, and thus, the present invention can contribute to the treatment of nerve damage or the regeneration of a nerve. The above-described composition of the present invention can be preferably used for introducing at least 1 gene selected from the group consisting of SOX10 gene and KROX20 gene or an expression product thereof.

The preparation method of the present invention is understood to encompass, in addition to direct reprogramming in vitro (in vitro), direct reprogramming in vivo (in vivo) as described above.

Direct reprogramming in vivo (in vivo) in addition to introduction of at least 1 gene selected from the group consisting of SOX10 gene and KROX20 gene or an expression product thereof into fibroblasts at the injured part of the nerve, direct reprogramming of cardiomyocytes in vivo (in vivo) as described in the following documents can be performed, for example.

1:Ieda M.Heart regeneration using reprogramming technology.Proc Jpn Acad Ser B Phys Biol Sci.2013；89(3):118-28.Review.

2:Ieda M,Fu JD,Delgado-Olguin P,Vedantham V,Hayashi Y,Bruneau BG,Srivastava D.Direct reprogramming of fibroblasts into functional cardiomyocytes by defined factors.Cell.2010Aug 6；142(3):375-86.

3:Qian L,Huang Y,Spencer CI,Foley A,Vedantham V,Liu L,Conway SJ,Fu JD,Srivastava D.In vivo reprogramming of murine cardiac fibroblasts into induced cardiomyocytes.Nature.2012May 31；485(7400):593-8.

The contents of the above-mentioned documents 1 to 3 are incorporated by reference in the present specification.

[ examples ] A method for producing a compound

The following examples are given by way of illustration only, and the present invention is not limited to these examples.

In the examples, HDF represents Human skin fibroblasts (Human Dermal fibroblasts). cSC shows cultured Schwann cells (cultured Schwann cells) collected from a control organism. dSC shows Schwann cells (direct reprogrammed Schwann cells) obtained by the method of the present invention.

Cont denotes Control.

Example 1 outline of the method (FIG. 1)

FIG. 1 shows an outline of a method for preparing Schwann cells ("directly reprogrammed Schwann cell: dSC" in the figure).

Puro was inserted into the retroviral vector plasmid pmxs using the Gene art system with the cDNA coding sequence of various genes such as SOX 10. Plat GP packaging cells were suspended in 1% NEAA 10% FBS DMEM (normal medium) containing 100U/mL penicillin and 100. mu.g/mL streptomycin in 10cm gelatin-coated culture plates at 5X 10 cells per plate⁶The seeds were sown at individual concentrations (day-3). After 24 hours of culture, pMXs vectors containing various genes were introduced in various combinations together with pCMV VSV vectors at the following ratios using X-tremeGENE 9. That is, a mixture of 5. mu.g of the gene to be introduced, 2.5. mu.g of pCMV. VSV, 500. mu.l of Opti-MEM, and 922.5. mu.l of X-tremeGENE was added to a 10cm plate containing 10ml of the medium (day-2). After 24 hours, the medium was changed to a normal medium containing no antibiotics (day-1). On the same day (day-1), a human normal skin fibroblast cell line (aHDF) ("fibroplast" in the figure) was cultured at 1.5X 10⁴～2×10⁴cells/mL were seeded in culture plates or 12-well plates. After 24 hours (day 0), Plat GP culture supernatant was passed through a 0.45 μm pore diameter syringe filter and mixed with polybutene (final concentration 4 μ g/mL) (virus solution). After removing the culture supernatant of aHDF by suction, 1mL of the above virus solution was added rapidly, and the mixture was cultured for 24 hoursSometimes (infection; in the figure, "transfection"). As a control group, cells not infected with the virus were also prepared. After 1 day (day 1), the culture supernatant was aspirated off, and schwann cell induction medium (medium with 5mM forskolin,10ng/ml recombiant human basic fibroblast growth factor (bFGF),5ng/ml recombiant human platelet-derived growth factor (PDGF) and 200ng/ml recombiant human heregulin1-b1(GGF) (both final concentrations) was added to the medium) was added, after which, new medium of the same composition was exchanged every 2 days. On days 12 to 22, S100. beta. staining was performed on the obtained cells as a representative marker of Schwann cells. The cells cultured in the same manner without infecting them with the retrovirus vector were designated as Control.

Example 2 conversion of human Normal skin fibroblasts into Schwann cells, S100 beta fluorescence Immunochromatographic image (Table 1) (FIG. 2)

Human normal skin fibroblasts (aHDF) were cultured in 12-well plates and subjected to the procedure described in example 1. On day 14, the culture medium was aspirated from each well, washed 1 time with PBS, and fixed with 4% PFA. The blocking solution was added and allowed to stand at 37 ℃ for 15 minutes. Cells stained with anti-S100 β antibody (primary antibody) and AlexaFluor566 labeled anti-rabbit IgG antibody (secondary antibody) were used. Combinations of different genes were introduced into each well of the plate, and combinations of genes into which number of wells were introduced are shown in table 1 (in the table, the column of each gene contains a substance indicated as "1" indicating that a retroviral vector containing the gene was infected, and the blank column indicates that a retroviral vector containing the gene was not infected). The gene candidates for the introduction of SOX10, Krox20, and Oct6, which are genes involved in Schwann cells, were 7 factors of SOX2, C-myc, KLF4, and Oct3/4, which are genes involved in reprogramming. For example, No.43 in Table 1 is a cell infected with a retroviral vector containing the genes of SOX10, Krox20, Oct6, KLF 4.

FIG. 2 shows representative staining patterns of S100. beta. cells into which the 2-factor genes of Sox10 and Krox20 were introduced. FIG. 2 shows co-staining with nuclear staining caused by DAPI (4', 6-diaminodino-2-phenylindole).

[ TABLE 1 ]

Example 3 conversion of human normal skin fibroblasts into Schwann cells and semi-quantitation of S100 β staining (FIG. 3) 3)

In the same experiment as in FIG. 2, in order to confirm the staining properties of other combinations, the plates were observed with a fluorescence microscope (manufactured by Olympus), and the staining properties of S100. beta. were evaluated in 4 stages (i.e. + + + +, -, in the order of S100. beta. positive cells).

Typical images of the respective evaluations are shown in fig. 3. The evaluation results are also shown in table 1. For example, it can be known that: cells infected with retroviral vector containing the 2 genes of SOX10 and KROX20 of No.4 were evaluated to be +++ and contained the most S100. beta. positive cells. Even though SOX10 alone or KROX20 alone can induce schwann cells at a low efficiency, co-introduction of SOX10 and KROX20 can induce them more efficiently. In addition, it can be seen that: oct6 had little effect on the induction of schwann cells.

Example 4 transformation of human Normal skin fibroblasts into Schwann cells and evaluation of cell morphology (FIG. 4)

In example 1, the cell morphology change in the transition from human normal skin fibroblasts to schwann cells in cells into which the 2-factor genes of Sox10 and Krox20 were introduced was evaluated as an increase in cell length/cell width ratio relative to the bipolar cell length characteristic of schwann cells.

The evaluation results are shown in fig. 4. The transformation revealed an increase in the ratio of cell length to cell width, i.e., a change to a cell morphology characteristic of Schwann cells.

Example 5 transformation of human Normal skin fibroblasts into Schwann cells with other Schwann cells Immunity of associated markersCellular fluorescence staining image (FIG. 5)

Human normal skin fibroblasts aHDF were cultured in 12-well plates and subjected to the procedure of example 1. The results of the 2 gene groups introduced with SOX10 and Krox20 are shown. 12 days after gene transfer, immunostaining was performed.

The results are shown in FIG. 5. The hosts of the anti-p 75NTR antibody (primary antibody), anti-GFAP antibody (primary antibody), and anti-NG 2 antibody (primary antibody) were rabbits, and the hosts of the anti-Nestin antibody (primary antibody) were mice. The secondary antibody was labeled with either AlexaFluor488,566 anti-rabbit IgG antibody or AlexaFluor488 labeled anti-mouse IgG antibody. Although the positive rate was decreased compared to S100 β, the expression of each was confirmed.

Example 6

A. Conversion of human Normal skin fibroblasts into Schwann cells, measurement of mRNA expression levels of S100. beta. and p75NTR genes (FIG. 6)

Human normal skin fibroblasts (aHDF) were cultured in 12-well plates and subjected to the procedure of example 1. The 2-factor genes of Sox10 and Krox20 were introduced into the cells of wells No.1 to 6, and no gene was introduced into the cells of wells No.7 to 12, as a control. 12 days after gene transfer, total RNA was recovered from each well with ISOGEN II, and cDNA was synthesized using a ReverTra Ace qPCR RT Master Mix. Real-time PCR Master Mix, Taqman probe, Specific Primer and cDNA were mixed for the purpose of quantifying mRNA levels of UCP1 and β actin genes, and Real-time RT-PCR was performed using AB7300Real-time PCR system. The value of the mRNA level of the gene of interest relative to the β actin mRNA level was calculated for each cell.

The results are shown in FIG. 6. The ordinate of the graph shows the value of well No.1 as "1" and the relative value of the calculated values of the respective wells as the relative mRNA level. Cells (Nos. 1 to 6) into which 2 genes of SOX10 and KROX20 were introduced showed strong expression of S100. beta. and p75NTR genes as cell-specific markers of Schwann as compared with control (Nos. 7 to 12).

Electrophoresis of RT-PCR products Using agarose gels

The Real-time RT-PCR product obtained in A was subjected to electrophoresis using an agarose gel. As a result, strong expression of mRNA of S100. beta. and p75NTR genes was confirmed in the cell population into which the 2-factor genes of SOX10 and Krox20 were introduced.

C. The use of RTPCR for direct reprogramming efficiency of human fibroblasts into schwann cells by combinations of no factor introduction, only SOX10, only Krox20, and SOX10+ Krox20, was studied.

aHDF, which is a human normal skin fibroblast, was cultured in a 12-well plate, and the procedure of example 1 was performed. In the same manner as in A, the expression of mRNA of S100. beta. and p75NTR was compared in cells in which no gene was introduced (control), only SOX10 gene was introduced, only Krox20 gene was introduced, and SOX10+ Krox20 gene was introduced. The relative value of mRNA is expressed as relative mRNA level. Values for the mRNA levels of the gene of interest relative to the β actin mRNA level were calculated for each group. The value of 1 group in non-cell-introduced cells was defined as "1", and the average of the relative values of the calculated values of the respective groups was expressed as the relative mRNA level. As a result, in the group into which the factor 2 of SOX10+ Krox20 had been introduced, the expression levels of mRNA for S100. beta. and p75NTR were greatly increased. Even in the case of cells into which only Sox10 had been introduced, the expression levels of mRNA of S100 β and p75NTR were increased, although the expression levels were lower than those of cells into which both Sox10 and Krox20 had been introduced. Even when the cell into which only Krox20 was introduced was lower than the cell into which both Sox10 and Krox20 were introduced, the mRNA expression level of S100 β was increased. Relative mRNA levels for S100 β were 4.4 in non-transfected cells, 1712.3 in Sox 10-introduced cells alone, 25.1 in Krox 20-introduced cells alone, and 127615.0 in Sox10+ Krox 20-introduced cells. The relative mRNA levels of p75NTR were 3.1 in non-transfected cells, 74.3 in Sox10 introduced into cells alone, 0.4 in Krox20 introduced into cells alone, and 35397.6 in Sox10+ Krox20 introduced cells. Therefore, even though SOX10 alone or Krox20 alone is inefficient, direct reprogramming from human fibroblasts to schwann cells may occur.

From the above, it was confirmed that: direct reprogramming of Schwann cells from human fibroblasts was efficient using factor 2 of SOX10 and Krox 20. In addition, it was shown that even with any factor 1 of SOX10 or Krox20, the efficiency is low but direct reprogramming of schwann cells by human fibroblasts is possible.

Example 7 study of neurite elongation Effect of 7 dSC on nerve cells (FIG. 7)

NG108-15 was cultured for 12 hours, followed by co-culture with HDF, cSC, or dSC (dSC induced by introduction of the 2-factor gene of Sox10 and Krox 20) for 36 hours. As a control, NG108-15 was cultured by DMEM supplemented with 1% FBS only. The ratio of the number of neurite-bearing cells (percent of neurite-bearing cells), the number of primary neurites directly elongated by the cell body (number of primary neurons), and the length of the Longest neurite (longerturbitute length) were determined by: tomita K, Madura T, Sakai Y, et al, Global identification of human adopside-derived stem cells, experiments for cell-based translation therapy. neuroscience.2013; 236:55-65. the comparative study was conducted. Only NG108-15 was fluorescently stained using anti-Tuj-1 antibody (primary antibody) and a secondary antibody AlexaFluor566 labeled anti-rabbit IgG antibody.

Fig. 7 shows the results. Both the cssc and the dSC showed high neurite outgrowth-promoting effects in all parameters (I-III) studied by comparison with the control group, and the performance of dSC was comparable to cSC. From the above, it was confirmed that: dSC the culture supernatant promotes process elongation for nerve cells.

Example 8 fibroblasts from normal skin of human being caused by introduction of Plasmid (electroporation) in a virus-free manner Conversion to Schwann cells (FIG. 8)

Human normal skin fibroblasts aHDF were cultured in 12-well plates and subjected to the procedure of example 1. The results of the 2 gene groups introduced with SOX10 and Krox20 are shown. 14 days after gene transfer, immunostaining was performed.

The results are shown in FIG. 8. The hosts for the anti-S100 β antibody (primary antibody), anti-p 75NTR antibody (primary antibody), and anti-GAP 43 antibody (primary antibody) were rabbits, and the secondary antibody was labeled with AlexaFluor566 as an anti-rabbit IgG antibody. Although the positive rate was lower than that in the case of using a retrovirus vector, the expression of each of S100 β, p75NTR, and GAP43 was confirmed, and the conversion from fibroblast aHDF to schwann cells was confirmed.

Example 9 transformation of human Normal adipose-derived Stem cells into Schwann cells with other Schwann cells Immunocyte fluorescent stained image of cell-associated marker (FIG. 9)

The transition from adipose-derived stromal cells to dSC was verified.

Method for

Mesenchymal cells derived from normal human fat (ADSCs) were cultured in 12-well plates, and the procedure of example 1 was performed. The results of the 2 gene groups introduced with SOX10 and Krox20 are shown. 14 days after gene transfer, immunostaining was performed.

Results

The results are shown in FIG. 9. The hosts of the anti-S100 β antibody (primary antibody), the anti-p 75NTR antibody (primary antibody), the anti-GAP 43 antibody (primary antibody), and the anti-Protein Zero antibody (primary antibody) were rabbits, and the secondary antibody was labeled with an anti-rabbit IgG antibody using AlexaFluor 566. In the staining with S100 β, about 40% of the cells showed positive. Although the positive rate was decreased compared to S100 β for other schwann cell markers, the expression of each marker was also confirmed. Protein Zero as a myelin marker also showed positive.

Specifically, as a result of inducing Schwann cells in the same manner as fibroblasts (fibroplasts), the morphology of the cells changed from that of mesenchymal cells derived from human normal fat to that of bipolar or nonpolar cells having a relatively small nucleus typical of Schwann cells. Furthermore, cells that changed to a typical cell morphology in schwann cells were positive (about 40-50%) for the schwann cell marker (S100 β) (fig. 9A).

In addition, the induced cells were positive for schwann cell marker (S100 β, GAP43), undifferentiated schwann cell marker (P75NTR), and myelin marker (P0) (fig. 9B).

Example 10 transformation of umbilical cord vascular endothelial cells into Schwann cellsThe transformation and use of (2) have other Schwann cell associations Fluorescent staining of immunocytes with labels (FIG. 10)

The transition from vascular endothelial cells to dSC was verified.

Method for

Umbilical vascular Endothelial Cells (Huvec: Human Umbilical vessel Endothelial Cells) were cultured in 12-well plates and subjected to the procedure of example 1. The results of the 2 gene groups introduced with SOX10 and Krox20 are shown. 14 days after gene transfer, immunostaining was performed.

Results

The results are shown in FIG. 10. The hosts of the anti-S100 β antibody (primary antibody), the anti-p 75NTR antibody (primary antibody), the anti-GAP 43 antibody (primary antibody), and the anti-Protein Zero antibody (primary antibody) were rabbits, and the secondary antibody was labeled with 566-labeled anti-rabbit IgG antibody. In the staining with S100 β, about 30% of the cells showed positive. Although the positive rate was decreased compared to S100 β for other schwann cell markers, the expression of each marker was also confirmed. Protein Zero as a myelin marker also showed positive.

Specifically, as a result of inducing Schwann cells in the same manner as fibroblasts (fibroplasts), the morphology of the cells changed from that of umbilical cord vascular endothelial cells to that of bipolar or nonpolar cells having a relatively small nucleus typical of Schwann cells. Furthermore, cells that changed to a typical cell morphology in schwann cells were positive (about 50-60%) for the schwann cell marker (S100 β) (fig. 10A).

The induced cells were positive for schwann cell markers (S100 β, GAP43) and undifferentiated schwann cell marker (p75NTR) (fig. 10B).

Example 11 dSC (dSC induced by introducing the 2-factor gene of Sox10 and Krox 20) and labeled with GFP Co-culture of DRGn resulted in dSC formation of myelin in vitro. Immunization using Schwann cell-associated markers Cell fluorescent dye image (FIG. 11)

Co-culture of dSC and DRGn with GFP was used to assess the in vitro myelin-forming ability of dSC.

Method for

dSC were cell-labeled with GFP using a retroviral vector.

The specific method is as follows: yoshioka T, Ageyama N, Shibata H, Yasu T, Misawa Y, Takeuchi K, Matsui K, Yamamoto K, Terao K, Shimada K, Ikeda U, Ozawa K, Hanazono Y.repair of immortal media transferred bone marrow-derived CD34+ stem cells in animal husman primate model.stem Cel.2005 Mar; 23(3) 355-64, and the literature: hirschmann F, Verhoeyen E, Wirth D, Bauwens S, Hauser H, Rudert m. viral marking of architectural chlorine cells by retroviral infection using green fluorescence protein. osteo identification cartilage.2002feb; 10(2) 109-18.

Posterior root ganglion cells (DRGn: dorsal root ganglion neuron) collected from postnatal day 5 mice were cultured in 12-well plates for 2 days, and were co-cultured in myelin differentiation-inducing medium containing dSC labeled with GFP, nerve growth factor, ascorbic acid and cAMP. Immunostaining was performed 14 days after the start of co-cultivation of the genes.

The composition of the medium used was as follows:

DMEM containing N2supplement(Invitrogen),50ng/ml ascorbic acid(Wako,Osaka,Japan),and 50ng/ml recombinant rat b-nerve growth factor(NGF)(R&D Systems,Inc.,Minneapolis,MN,USA),0.5μM cAMP(R&D Systems,Inc.,Minneapolis,MN,USA)。

the culture medium was as described in the literature: sango K, Kawakami E, Yanagisawa H, Takaku S, Tsukamoto M, Utsunomiya K, Watabe K.Myelination in culture of estableshed neuronal and Schwann Cell lines.Histochem Cell biol.2012Jun; 137(6) 829-39.

The host of the anti-neurofilament antibody (primary antibody) and the anti-MBP (Myelin Basic Protein) antibody (primary antibody) is a mouse, the host of the anti-Tuj-1 antibody (primary antibody) and the anti-Protein Zero antibody (primary antibody) is a rabbit, and the secondary antibody is an IgG antibody of AlexaFluor566 labeled anti-mouse and rabbit, and an IgG antibody of Cy5 labeled anti-mouse and rabbit. anti-MBP antibody and anti-Protein Zero antibody as myelin marker antibody were labeled with red, and anti-neurofilament antibody and anti-Tuj-1 antibody as neuroaxon marker were labeled with gray.

Results

The results are shown in FIG. 11. The myelin-forming Schwann cells were visible along the neurites of the DRG, but a portion of the myelin-forming Schwann cells overlapped with dSC cells labeled with GFP.

Example 12 inhibition of dSC labeled with GFP (2-cause introduced Sox10 and Krox 20) against sciatic nerve injury model dSC induced by daughter gene) transplantation. Immunization with myelin-associated markers Epidemic cell fluorescence staining image (FIG. 12)

dSC was investigated for its ability to phosphorylate myelin in vivo.

Method for

dSC were cell-labeled with GFP using viral vectors.

The sciatic nerve of the immunodeficient mice was exposed, and the central portion was held with surgical hemostats for about 1 minute to crush the nerves within a range of about 5 mm. About 5 ten thousand of dSC labeled with GFP were injected to the distal side. 1 month after the start of transplantation, the repair nerves of the crushed parts were collected and immunostained. Fig. 12 shows an outline of the method.

The host of the anti-neurofilament antibody (primary antibody) and the anti-MBP antibody (primary antibody) was a mouse, the host of the anti-Tuj-1 antibody (primary antibody) and the anti-Protein Zero antibody (primary antibody) was a rabbit, and the secondary antibody was an IgG antibody against mouse and rabbit labeled with AlexaFluor566, and an IgG antibody against mouse and rabbit labeled with Cy 5. anti-MBP antibody and anti-Protein Zero antibody as myelin marker antibody were labeled with red, and anti-neurofilament antibody and anti-Tuj-1 antibody as neuroaxon marker were labeled with gray.

Results

The results are shown in FIG. 12. GFP + cells were aligned in a manner to follow the regenerating nerve, and these overlapped with myelin marker + cells (fig. 12B and C).

GFP + cells that were unstable to (erratic parasitism, Japanese: adduction) regenerated nerves also expressed the myelin marker (FIGS. 12B and C, arrows).

EXAMPLE 13 transplantation of dSC to ischial Defect (5mm) model of immunodeficient mice (FIG. 13)

Method for

Cultured Schwann cells (SC: comparative control group) were isolated from the sciatic nerve and cultured. These cells were seeded in the gelatin tube in advance, and a notch of about 5mm was made in the sciatic nerve trunk of the mouse. The obtained mixed tube was transplanted to the nerve defect portion. The nerve regeneration-promoting effect was evaluated using Sham mice and a tube-transplanted group (Cont) containing only PBS as a control (fig. 13A).

Reconstructed nerves show macroscopic images and myelin-stained images caused by the lux fast blue of cross-axial sections of regenerated nerve tissue. Sciatic nerve function showed results after 6 weeks (6W) and 12 weeks (12W) after transplantation, and the contraction of the innervating muscles was evaluated as wet muscle weight.

The Sciatic nerve function Index (SFI: scientific functional Index) was as per literature: inserra MM, Bloch DA, terrris DJ.functional indices for scientific, permanent, and spatial neural mutations in the mouse, Microspur.1998; 18:119 and 124.

Atrophy and fibrosis of the innervated muscles were according to the literature: Clavijo-Alvarez JA, Nguyen VT, Santiago LY, Doctor JS, Lee WP, Marra KG. Comparison of biocidable compositions with diagnostic novel defects. plant Reconster Surg.2007; 119: 1839-.

Results

In the reconstructed macroscopic image of the nerve, both cSC groups and dSC were superior to control, and no significant difference was observed between the two (fig. 13B).

Among myelin-stained images of transverse sections of regenerated nerve tissue, dSC groups were comparable to cSC groups (fig. 13C).

In the Sciatic nerve function Index (SFI: scientific functional Index), dSC groups were able to see a recovery of Sciatic nerve function comparable to cSC at time 12W (FIG. 13D).

Significant differences from controls were also seen in both groups with respect to dominant muscle atrophy and fibrosis, but no significant difference between cSC and dSC was seen (fig. 13E).

Example 14 transformation into Schwann cells (Table 2)

The same experiment as in example 2 was performed for combinations of genes shown in Table 2.

Combinations of genes to be introduced into wells of which number are shown in Table 2 (in the table, the column of each gene contains a substance indicated as "1" indicating that a retroviral vector containing the gene is to be infected, and the blank column indicates that a retroviral vector containing the gene is not to be infected).

The staining property of S100. beta. was evaluated in 4 stages (i.e., in order of +++, ++, and, -, where the substance is abundant in S100. beta. positive cells) by observing the plate with a fluorescence microscope (manufactured by Olympus) in the same manner as in example 3.

The evaluation results are shown in table 2.

From the results, it is found that: oct6 is known as a factor that performs an important function in the differentiation of schwann cells, but it hardly induces direct reprogramming of schwann cells by somatic cells. In addition, it can be seen that: oct6 failed to improve the efficiency of direct reprogramming of schwann cells by somatic cells induced by Sox10 alone, Krox20 alone, or Sox10+ Krox 20.

[ TABLE 2-1 ]

[ TABLE 2-2 ]

Example 15 production of neurotrophic factors (FIG. 14)

HDF, cSC, and dSC were adjusted to 4X 10⁴cells/cm²Under the conditions of (1) seeding in cell cultureAfter confirming 80% confluence with the plate, the cells were cultured in the medium for cell supernatant collection for 48 hours. Thereafter, the culture supernatant of each group was passed through a 40- μm filter and collected. For Brain-derived Neurotrophic Factor (BDNF), glial cell line-derived Neurotrophic Factor (GDNF), and Brain Neurotrophic Factor (Nerve Growth Factor (NGF)), ELISA kits human BDNF, GDNF, and NGF (Promega, Madison, WI) were used to measure the amount of protein contained in the culture supernatant of each cultured cell.

As a result, both cSC and dSC produced all BDNF, GDNF, NGF more strongly than control (HDF). Additionally, BDNF production was similar even at the highest point, cSC and dSC.

Claims (5)

1. A method for producing Schwann cells directly from somatic cells without the intervention of pluripotent stem cells, comprising the step of introducing a combination of the SOX10 gene or its expression product and the KROX20 gene or its expression product into mammalian somatic cells.

2. The method of claim 1, wherein,

the somatic cell is a fibroblast, a vascular endothelial cell or a mesenchymal stem cell.

3. A graft material for use in the treatment of a nerve-based defect or a defect, deficiency or reduced function disease of Schwann cells, comprising cells obtained by the method of claim 1 or 2,

the disease is selected from multiple sclerosis, the spinal cord inflammation (Devic syndrome), concentric sclerosis (Balo disease), Acute Disseminated Encephalomyelitis (ADEM), inflammatory wide sclerosis (Schilder disease), Subacute Sclerosing Panencephalitis (SSPE), Progressive Multifocal Leukoencephalopathy (PML), Guillain-Barre syndrome, Fisher syndrome, chronic inflammatory demyelinating radiculitis, and peroneal muscular atrophy (CMT).

4. Use of the SOX10 gene or expression product thereof and the KROX20 gene or expression product thereof for the manufacture of a composition for preparing schwann cells from somatic cells.

5. Use of Schwann cells obtained by introducing a SOX10 gene or an expression product thereof and a KROX20 gene or an expression product thereof into somatic cells of a mammal, for producing a therapeutic agent for a disease caused by a defect in nerves or a defect, deficiency, or reduced function of Schwann cells,

the disease is selected from multiple sclerosis, the spinal cord inflammation (Devic syndrome), concentric sclerosis (Balo disease), Acute Disseminated Encephalomyelitis (ADEM), inflammatory wide sclerosis (Schilder disease), Subacute Sclerosing Panencephalitis (SSPE), Progressive Multifocal Leukoencephalopathy (PML), Guillain-Barre syndrome, Fisher syndrome, chronic inflammatory demyelinating radiculitis, and peroneal muscular atrophy (CMT).

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